Hla alleles associated with adverse drug reactions and methods for detecting such

ABSTRACT

This invention relates to a method of determining the presence of certain HLA alleles, such as HLA-B*1502 or HLA-B*5801, and a kit for carrying out this method. Also disclosed is a method for assessing whether a patient is at risk for developing adverse drug reactions (e.g., Stevens-Johnson syndrome, toxic epidermal necrolysis, or hypersensitivity syndrome) based on the presence or absence of a genetic marker (e.g., HLA-B*1502, HLA-B*5801, or HLA-B*4601).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application No.60/800,121, filed May 11, 2006, the content of which is incorporatedherein by its entirety.

BACKGROUND

An adverse drug reaction (ADR) is an undesired and unintended effect ofa drug. In particular, an adverse drug reaction occurs at doses used forprophylaxis, diagnosis or therapy. According to a widely citedmeta-analysis, ADRs were ranked between the fourth and sixth most commoncause of death (Lazarou et al., JAMA, 279(15):1200-1205, 1998).Cutaneous ADRs account for about 2-3% of all hospital admissions (Bigbyet al., JAMA, 256(24):3358-3363, 1986). They range from mildmaculopapular (MPE), with increasing severity, to life-threatening ADRs,such as hypersensitivity syndrome (HSS), Stevens-Johnson syndrome (SJS)and toxic epidermal necrolysis (TEN; Lyell's syndrome). The mortalityrate of the latter can be as high as 40%.

HSS is characterized by multi-organ involvements (e.g. hepatitis ornephritis) accompanied with systemic manifestations (e.g. fever,arthragis, eosinophilia and lymphadenopathy) in additional to skinrashes (Roujeau et al., N. Engl. J. Med., 331:1272-1285, 1994). SJS andTEN are immune-complex-mediated hypersensitivity disorders characterizedby rapid development of blistering exanthema of purpuric macules andtarget-like lesions accompanied with mucules involvement and skindetachments (Roujeau et al. J Invest Dermatol, 1994, 102:28S-30S). Theyare caused mostly by drugs, such as sulfonamides, anticonvulsants,allopurinol, nonsteroidal anti-inflammatory drugs, and antimalarials(Roujeau et al., N. Engl. J. Med., 333(24):1600-1607, 1995). In Taiwan,anticonvulsants (e.g., CBZ, phenytoin and phenobarbital) and allopurinolare the most common drugs causing SJS/TEN.

Recent developments of pharmacogenomics have implied that thesusceptibility to ADRs is associated with particular genetic alleles.For example, genomic polymorphisms of the thiopurine methyltransferasegene were found to be closely related to ADRs induced by azathioprine, adrug for rheumatologic diseases or cancer (Yates et al., Ann. Intern.Med., 126(8):608-614, 1997). It is also suggested that thesusceptibility to SJS/TEN/HSS induced by certain drugs is geneticallydetermined (Gennis M A, Am. J. Med., 91(6):631-634, 1991; Edwards S G,Postgrad. Med. J., 75(889):680-681, 1999). However, the exactresponsible genetic factors have yet to be identified.

These pharmacogenomics studies suggest that detecting ADR-associatedalleles in a patient is a useful approach for assessing whether thatpatient is at risk for developing ADRs. This kind of moleculardiagnostics certified by Clinical Laboratory Improvement Amendments isnow offered by reference laboratories in the US and Europe.

To determine the presence of a particular genetic allele, one or moreallelic-specific nucleotide need to be detected. In many cases, multipleregions within the allele must be targeted to achieve an accuratedetermination. For example, currently available methods for determiningan HLA-B allele (e.g., HLA-B*1502 or HLA-B*5801) requires detecting atleast 6 regions within that allele.

SUMMARY OF THE INVENTION

The present invention provides a method of predicting the risk of apatient for developing adverse drug reactions, particularly SJS, TEN, orHSS. It was discovered that HLA-B*1502 is associated with SJS/TENinduced by a variety of drugs, e.g., carbamazepine (CBZ). In addition,HLA-B*5801 is particularly associated with SJS/TEN induced byallopurinol. HLA-B*5801 is also associated with allopurinol-induced HSS.Milder cutaneous reactions induced by CBZ, such as maculopapular rash,erythema multiforme, urticaria, and fixed drug eruption, areparticularly associated with HLA-B*4601.

Accordingly, the present application provides a method of assessing therisk of a patient for developing an adverse drug reaction in response toa drug, including performing HLA typing using a biological sample fromthe patient. Any HLA allele that is associated with the ADR with asensitivity of at least about 40% can be used as the risk factor in thepresent invention. Preferably, the sensitivity of the risk factor is atleast about 50%, 60%, 70%, 80%, 85% or 90%. More preferably, thesensitivity is at least 95%. The drug is preferably selected from thegroup consisting of CBZ, oxcarbazepine (brand name: trileptal),licarbazepine, allopurinol, phenytoin, sulfasalazine, amoxicillin,ibuporfen and ketoprofen. Alternatively, the drug is preferably not anonsteroidal anti-inflammatory drug. Preferably, an HLA-B allele is therisk factor.

Assessing the risk of a patient for developing an adverse drug reactionin response to a drug, can be accomplished by determining the presenceof an HLA-B allele selected from the group consisting of HLA-B*1502,HLA-B*5801 and HLA-B*4601, wherein the presence of the HLA-B allele isindicative of a risk for an adverse drug reaction. The drug can beselected from the group consisting of CBZ, oxcarbazepine, licarbazepine,allopurinol, oxypurinol, phenytoin, sulfasalazine, amoxicillin,ibuprofen, and ketoprofen.

The adverse drug reaction can be a cutaneous adverse drug reaction, suchas SJS, TEN, or HSS. In one embodiment, the drug is selected from CBZ,oxcarbazepine, licarbazepine, and the allele is HLA-B*1502. In anotherembodiment, HLA-B*5801 is used to predict the risk for SJS, TEN, or HSS,and the drugs can be selected from allopurinol or oxypurinol. Othersubtypes of HLA-B15, B58 or B46, such as HLA-B*1503 or *1558, can alsobe used to predict the risk for developing an ADR.

A genetic allele can be detected by direct detection ofregions/nucleotides within the allele using genomic DNAs prepared frombiosamples, e.g., blood, saliva, urine or hair. The allele can also bedetected by, for example, serological or microcytotoxicity methods. Italso can be determined by detecting an equivalent genetic marker of theallele, which can be, e.g., an SNP (single nucleotide polymorphism), amicrosatellite marker or other kinds of genetic polymorphisms. In otherwords, the presence of the HLA-B*1502, 5801 or 4601 haplotype, ratherthan the allele per se, is indicative of a risk for adverse drugreactions. Exemplary equivalent genetic markers of HLA-B B*1502haplotype include DRB1*1202, Cw*0801, Cw*0806, A*1101, and MICA*019.Exemplary equivalent genetic markers of HLA-B*5801 includes HLA-A*3303,Cw*0302, DRB1*0301, and MICA*00201.

Another aspect of the present invention is a method of pharmacogenomicsprofiling. This method includes determining the presence of at least oneHLA-B allele selected from the group consisting of HLA-B*1502,HLA-B*5801, and HLA-B*4601. In one example, the presence of at least twoalleles selected from the group is determined, such as HLA-B*1502 andHLA-B*5801. In another example, the presence of all three alleles isdetermined. The method can optionally comprise the determination ofother genetic factors. Those other genetic factors can be associatedwith the predisposition for any disease or medical condition, includingADRs. For example, these other genetic factors can be selected from thegroup consisting of thiopurine methyltransferase and the genes for thelong-QT syndrome.

Also within the scope of this invention is a method for determiningwhether a patient carries HLA-B*1502 or HLA-B*5801. This method includesthe steps of: (1) detecting a first region selected from either Regions1-6 of HLA-B*1502 or Regions 1-6 of HLA-B*5801, (2) detecting a secondregion selected from either Regions 1-6 of HLA-B*1502 or Regions 1-6 ofHLA-B*5801, the second region being different from the first region, and(3) determining whether the patient carries the allele of interest, thepresence of the first and second regions indicating that the patientcarries HLA-B*1502 or HLA-B*5801. These regions can be detected byReal-Time PCR or Competitive Sequence-Specific Oligonucleotidehybridization assay coupled with ELISA (CSSO-ELISA). In one example,detecting two regions selected from Regions 1-6 of HLA-B*1502 or fromRegions 1-6 of HLA-B*5801 is sufficient to determine the presence orabsence of HLA-B*1502 or HLA-B*5801. Alternatively, three or moreregions within HLA-B*1502 or HLA-B*5801 are detected.

Detection of Region 1, Region 2, and Region 3 of HLA-B*1502 can beachieved respectively by identifying nucleotides at positions 1 and 3within Region 1, at positions 1 and 6 within Region 2, and at positions1 and 3 within Region 3 (including the nucleotides in either the sensestrand or the anti-sense strand at these positions).

Detection of Regions 1-6 of HLA-B*5801 can be achieved respectively byidentifying nucleotides at positions 1, 2, and 5 within Region 1, atpositions 1, 4, 6, 7, 8, 15, 16, and 20 within Region 2, at positions 2,4, 5, 8, and 9 within Region 3, at positions 3 and 5 within Region 4, atpositions 1 and 9 within Region 5, and at positions 3 and 10 withinRegion 6.

The present invention also provides a kit for detecting a geneticallele, e.g., HLA-B*1502 or HLA-B*5801. In one example, this kitcontains a first probe and a second probe, each targeting a regionselected from Regions 1-6 of HLA-B*1502 or Regions 1-6 of HLA-B*5801.The two probes target different regions. The kit can further include athird probe that targets an internal control allele. It can also includeone or more additional probes for detecting one or more additionalregions within HLA-B*1502 or HLA-B*5801.

Further provided is a method for determining whether a compound is acandidate that induces an ADR (e.g., SJS/TEN, HSS, or a milder cutaneousADR) in a patient carrying an HLA allele (e.g., HLA-B*1502, HLA-B*5801,or HLA-B*4601) associated with the ADR induced by a drug (e.g., CBZ,allopurinol, or phenytoin). This method includes the steps of: (1)isolating T cells from an ADR patient carrying an HLA allele associatedwith the ADR, (2) expanding T cells reactive to the drug that inducesthe ADR, (3) isolating antigen-presenting cells from the patient, (4)contacting the expanded T cells with a compound in the presence of theAPCs, and (5) examining whether the compound activates the expanded Tcells. A compound that activates the T cells is a candidate that inducesthe ADR in a patient carrying the same HLA allele.

A patient has a “risk” for an ADR if the probability of the patient todevelop an ADR is higher than the probability of the general populationto develop the ADR. The probability of the patient to develop the ADR isat least about 1.5 fold, more preferably at least about 2 fold, stillmore preferably at least about 3, 4, 5, 6, 7, 8 or 9 fold, and mostpreferably at least about 10 fold as high as the probability of thegeneral population to develop the ADR. The probability can be determinedby any method known in the art, such as by using the incidence of riskfactors. For example, a given risk factor is present in 5% of thegeneral population. If this factor is present in 10% of the patients whohave an ADR, then the probability of a patient with this risk factor todevelop the ADR is 2 fold as high as the probability of the generalpopulation to develop the ADR.

A “risk factor” for an ADR is a factor that is associated with the ADR.The sensitivity of a risk factor is preferably at least about 40%, morepreferably at least about 50%, 60%, 70%, 80%, 85% or 90%. Mostpreferably, the sensitivity is at least 95%. The “sensitivity” of a riskfactor for predicting an ADR is the percentage of patients with the ADRthat possess the risk factor. In other words, if every SJS patient hasallele A, the sensitivity of allele A for predicting SJS is 100%. If 20out of 40 SJS patients have allele B, then the sensitivity of allele Bfor predicting SJS is 50%.

An “equivalent genetic marker” of an allele of interest refers to agenetic marker that is linked to the allele of interest, i.e., itdisplays a linkage disequilibrium with the allele of interest.

“Pharmacogenomics profiling” refers to the determination of geneticfactors present in a subject that are associated with diseases ormedical conditions, particularly adverse reactions to drugs. Typically,a panel of genetic factors is determined in pharmacogenomics profiling,and the factors may or may not be associated with the same disease,medical condition, or reaction to drug.

A drug compound, as used herein, refers to a compound that is a drug orthe same as the drug except that at least one hydrogen in the drug issubstituted with a halo, hydroxyl, acylamino, alkyl, alkenyl, alkynyl,alkoxy, aryloxy, aryl, aryloxyaryl, carboxyl, carboxylalkyl,carboxyl-substituted alkyl, carboxyl-cycloalkyl, carboxylsubstitutedcycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxylheteroaryl,carboxyl-substituted heteroaryl, carboxylheterocyclic,carboxyl-substituted heterocyclic, cycloalkyl, substituted alkyl,substituted alkoxy, substituted aryl, substituted aryloxy, substitutedaryloxyaryl, substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic or substituted heterocyclic group. The chemical groups aredefined below. The subsistent can contain zero to ten, zero to six, zeroto four, or zero to two carbon atoms.

As used herein, “alkyl” refers to alkyl groups having 1 to 10 carbonatoms or 1 to 6 carbon atoms. “Substituted alkyl” refers to an alkylgroup, preferably of from 1 to 10 carbon atoms, having from 1 to 5substituents selected from the group consisting of alkoxy, substitutedalkoxy, acyl, acylamino, thiocarbonylamino, acyloxy, amino, amidino,alkyl amidino, thioamidino, aminoacyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aryl, substituted aryl,aryloxy, substituted aryloxy, aryloxylaryl, substituted aryloxyaryl,cyano, halogen, hydroxyl, nitro, carboxyl, carboxyalkyl,carboxyl-substituted alkyl, carboxyl-cycloalkyl, carboxyl-substitutedcycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxylheteroaryl,carboxyl-substituted heteroaryl, carboxylheterocyclic,carboxylsubstituted heterocyclic, cycloalkyl, substituted cycloalkyl,guanidino, guanidinosulfone thiol, thioalkyl, substituted thioalkyl,thioaryl, substituted thioaryl, thiocycloalkyl, substitutedthiocycloalkyl, thioheteroaryl, substituted thioheteroaryl,thioheterocyclic, substituted thioheterocyclic, heteroaryl, substitutedaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic,cycloalkoxy, substituted cycloalkoxy, heteroaryloxy, substitutedheteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy,oxycarbonylamino, oxythiocarbonylamino, —OS(O)₂-alkyl,—OS(O)₂-substituted alkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl,—OS(O)₂-heteroaryl, —OS(O)₂-substituted heteroaryl,—OS(O)₂-heterocyclic, —OS(O)₂-substituted heterocyclic, —OSO₂—NRR,—NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl,—NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl, —NRS(O)₂-substitutedheteroaryl, —NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic,—NRS(O)₂—NR-substituted heterocyclic, mono- and di-alkylamino, mono- anddi-(substituted alkyl)amino, mono and di-arylamino, mono- anddi-(substituted aryl)amino, mono- and diheteroarylamino, mono- anddi-(substituted heteroaryl)amino, mono- and diheterocyclic amino, mono-and di-(substituted heterocyclic) amino, unsymmetric disubstitutedamines having different substituents selected from the group consistingof alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic;substituted alkyl groups having amino groups blocked by conventionalblocking groups and alkyl/substituted alkyl groups substituted with—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substitutedalkenyl, —SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-aryl,—SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substituted heteroaryl,—SO₂-heterocyclic, —SO₂-substituted heterocyclic or —SO₂—NRR, where R ishydrogen or alkyl.

“Alkoxy” refers to the group “alkyl-O—,” which includes, by way ofexample, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy,sec-butoxy, npentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.“Substituted alkoxy” refers to the group “substituted alkyl-O—.”

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O), cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—,substituted heteroaryl-C(O), heterocyclic-C(O)—, and substitutedheterocyclic-C(O)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

“Acylamino” refers to the group —C(O)NRR where each R is independentlyselected from the group consisting of hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic; andwhere each R can be joined to form, together with the nitrogen atom, aheterocyclic or substituted heterocyclic ring wherein alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic are asdefined herein.

“Acyloxy” refers to the groups alkyl-C(O)O—, substituted alkyl-C(O)O—,alkenyl-C(O)O—, substituted alkenyl-C(O)O—, alkynyl-C(O)O—, substitutedalkynyl C(O)O—, aryl-C(O)O—, substituted aryl-C(O)O—, cycloalkyl-C(O)O—,substituted cycloalkyl-C(O)O—, heteroaryl-C(O)O—, substitutedheteroaryl-C(O)O—, heterocyclic—C(O)O—, and substitutedheterocyclic—C(O)O— wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

“Alkenyl” refers to alkenyl group preferably having from 2 to 10 carbonatoms and more preferably 2 to 6 carbon atoms and having at least 1 andpreferably from 1-2 sites of alkenyl unsaturation. “Substituted alkenyl”refers to alkenyl groups having from 1 to 5 substituents selected fromthe group consisting of alkoxy, substituted alkoxy, acyl, acylamino,thiocarbonylamino, acyloxy, amino, amidino, alkylamidino, thioamidino,aminoacyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aryl, substituted aryl, aryloxy, substituted aryloxy, aryloxyaryl,substituted aryloxyaryl, halogen, hydroxyl, cyano, nitro, carboxyl,carboxylalkyl, carboxyl-substituted alkyl, carboxylcycloalkyl,carboxyl-substituted cycloalkyl, carboxylaryl, carboxyl-substitutedaryl, carboxylheteroaryl, carboxyl-substituted heteroaryl,carboxylheterocyclic, carboxyl-substituted heterocyclic, cycloalkyl,substituted cycloalkyl, guanidino, guanidinosulfone, thiol, thioalkyl,substituted thioalkyl, thioaryl, substituted thioaryl, thiocycloalkyl,substituted thiocycloalkyl, thioheteroaryl, substituted thioheteroaryl,thioheterocyclic, substituted thioheterocyclic, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy,substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy,heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino,oxythiocarbonylamino, —OS(O)₂-alkyl, —OS(O)₂-substituted alkyl,—OS(O)₂-aryl, —OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NRR, —NRS(O)₂-alkyl,—NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl, —NRS(O)₂-substituted aryl,—NRS(O)₂-heteroaryl, —NRS(O)₂-substituted heteroaryl,—NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-alkyl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic,—NRS(O)₂—NR-substituted heterocyclic; mono- and di-alkylamino, mono- anddi-(substituted alkyl)amino, mono and di-arylamino, mono- anddi-(substituted aryl)amino, mono- and diheteroarylamino, mono- anddi-(substituted heteroaryl)amino, mono- and di-heterocyclic amino, mono-and di-(substituted heterocyclic) amino, unsymmetric di-substitutedamines having different substituents selected from the group consistingof alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic andsubstituted alkenyl groups having amino groups blocked by conventionalblocking groups (such as Boc, Cbz, formyl, and the like) andalkenyl/substituted alkenyl groups substituted with —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substituted alkenyl,—SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-aryl,—SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substituted heteroaryl,—SO₂-heterocyclic, —SO₂-substituted heterocyclic or —SO₂ NRR, where R ishydrogen or alkyl.

“Alkynyl” refers to alkynyl group preferably having from 2 to 10 carbonatoms and more preferably 3 to 6 carbon atoms and having at least 1 andpreferably from 1-2 sites of alkynyl unsaturation. “Substituted alkynyl”refers to alkynyl groups having from 1 to 5 substituents selected fromthe group consisting of alkoxy, substituted alkoxy, acyl, acylamino,thiocarbonylamino, acyloxy, amino, amidino, alkylamidino, thioamidino,aminoacyl, aminocarbonylamino, aminothiocarbonylamino,arninocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryloxy,aryloxyaryl, substituted aryloxyaryl, halogen, hydroxyl, cyano, nitro,carboxyl, carboxylalkyl, carboxyl-substituted alkyl, carboxylcycloalkyl,carboxyl-substituted cycloalkyl, carboxylaryl, carboxyl-substitutedaryl, carboxylheteroaryl, carboxyl-substituted heteroaryl,carboxylheterocyclic, carboxyl-substituted heterocyclic, cycloalkyl,substituted cycloalkyl, guanidino, guanidinosulfone, thiol, thioalkyl,substituted thioalkyl, thioaryl, substituted thioaryl, thiocycloalkyl,substituted thiocycloalkyl, thioheteroaryl, substituted thioheteroaryl,thioheterocyclic, substituted thioheterocyclic, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy,substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy,heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino,oxythiocarbonylamino, —OS(O)₂-alkyl, —OS(O)₂-substituted alkyl,—OS(O)2-aryl, —OS(O)₂-substituted aryl, ⁻OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NRR, —NRS(O)₂-alkyl,—NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl, —NRS(O)₂-substituted aryl,—NRS(O)₂-heteroaryl, —NRS(O)₂-substituted heteroaryl,—NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic,—NRS(O)₂—NR-substituted heterocyclic, mono- and di-alkylamino, mono- anddi-(substituted alkyl)amino, mono and di-arylamino, mono- anddi-(substituted aryl)amino, mono- and diheteroarylamino, mono- anddi-(substituted heteroaryl)amino, mono- and di-heterocyclic amino, mono-and di-(substituted heterocyclic) amino, unsymmetric di-substitutedamines having different substituents selected from the group consistingof alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic;substituted alkynyl groups having amino groups blocked by conventionalblocking groups (such as Boc, Cbz, formyl, and the like), andalkynyl/substituted alkynyl groups substituted with —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substituted alkenyl,—SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-aryl,—SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substituted heteroaryl,—SO₂-heterocyclic, —SO₂-substituted heterocyclic or —SO₂NRR, where R ishydrogen or alkyl.

“Aminoacyl” refers to the groups —NRC(O)alkyl, —NRC(O)substituted alkyl,—NRC(O)cycloalkyl, —NRC(O)substituted cycloalkyl, —NRC(O)alkenyl,—NRC(O)-substituted alkenyl, —NRC(O)-alkynyl, —NRC(O)-substitutedalkynyl, —NRC(O)-aryl, —NRC(O)substituted aryl, —NRC(O)heteroaryl,—NRC(O)substituted heteroaryl, —NRC(O)heterocyclic, and—NRC(O)substituted heterocyclic, where R is hydrogen or alkyl andwherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Aminocarbonyloxy” refers to the groups —NRC(O)O-alkyl,—NRC(O)O-substituted alkyl, —NRC(O)O-alkenyl, —NRC(O)O-substitutedalkenyl, —NRC(O)O alkynyl, —NRC(O)O-substituted alkynyl,—NRC(O)O-cycloalkyl, —NRC(O)O substituted cycloalkyl, —NRC(O)O-aryl,—NRC(O)O-substituted aryl, —NRC(O)O heteroaryl, —NRC(O)O-substitutedheteroaryl, —NRC(O)O-heterocyclic, and —NRC(O)O-substituted heterocyclicwhere R is hydrogen or alkyl and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Oxycarbonylamino” refers to the groups —OC(O)NRR, —OC(O)NR-alkyl,—OC(O)NR-substituted alkyl, —OC(O)NR-alkenyl, —OC(O)NR-substituentalkenyl, —OC(O)NR-alkynyl, —OC(O)NR-substituted alkynyl,—OC(O)NR-cycloalkyl, —OC(O)NR-substituted cycloalkyl, —OC(O)NR-aryl,—OC(O)NR-substituted aryl, —OC(O)NR-heteroaryl, —OC(O)NR-substitutedheteroaryl, —OC(O)NR-heterocyclic, and —OC(O)NR-substituted heterocyclicwhere R is hydrogen or alkyl, and where each R can be joined to form,together with the nitrogen atom, a heterocyclic or substitutedheterocyclic ring and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Aryl” or “Ar” refers to an unsaturated aromatic carbocyclic group offrom 6 to 14 carbon atoms having a single ring (e.g., phenyl) ormultiple condensed rings (e.g., naphthyl or anthryl) which condensedrings may or may not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like). Preferred aryls includephenyl and naphthyl.

Substituted aryl refers to aryl groups which are substituted with from 1to 3 substituents selected from the group consisting of hydroxy, acyl,acylamino, thiocarbonylamino, acyloxy, alkyl, substituted alkyl, alkoxy,substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, amidino, alkylamidino, thioamidino, amino, aminoacyl,aminocarbonyloxy, aminocarbonylamino, aminothiocarbonylamino, aryl,substituted aryl, aryloxy, substituted aryloxy, cycloalkoxy, substitutedcycloalkoxy, heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy,substituted heterocyclyloxy, carboxyl, carboxylalkyl,carboxylsubstituted alkyl, carboxyl-cycloalkyl, carboxyl-substitutedcycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxylheteroaryl,carboxyl-substituted heteroaryl, carboxylheterocyclic,carboxyl-substituted heterocyclic, carboxylamido, cyano, thiol,thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl,thioheteroaryl, substituted thioheteroaryl, thiocycloalkyl, substitutedthiocycloalkyl, thioheterocyclic, substituted thioheterocyclic,cycloalkyl, substituted cycloalkyl, guanidino, guanidinosulfone, halo,nitro, heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkoxy, substituted cycloalkoxy, heteroaryloxy,substituted heteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy,oxycarbonylamino, oxythiocarbonylamino, —SO(O)₂-alkyl,—S(O)₂-substituted alkyl, —S(O)₂-cycloalkyl, —S(O)₂-substitutedcycoalkyl, —S(O)₂-alkenyl, —S(O)₂-substituted alkenyl, —S(O)₂-aryl,S(O)₂-substituted aryl, —S(O)₂-heteroaryl, —S(O)₂-substitutedheteroaryl, —S(O)₂-heterocyclic, —S(O)₂-substituted heterocyclic,—OS(O)₂-alkyl, —OS(O)₂-substituted alkyl, —OS(O)₂-aryl,—OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl, —OS(O)₂-substitutedheteroaryl, —OS(O)₂-heterocyclic, —OS(O)₂-substituted heterocyclic,—OSO₂—NRR, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl,—NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl, —NRS(O)₂-substitutedheteroaryl, —NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-substituted—NRS(O)₂—NR-substituted heterocyclic, mono- and di-alkylamino, mono- anddi-(substituted alkyl)amino, mono- and di-arylamino, mono- anddi-(substituted aryl)amino, mono and di-heteroarylamino, mono- anddi-(substituted heteroaryl)amino, mono- and di-heterocyclic amino, mono-and di-(substituted heterocyclic) amino, unsymmetric di-substitutedamines having different substituents selected from the group consistingof alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, substituted heterocyclic, aminogroups on the substituted aryl blocked by conventional blocking groups(such as Boc, Cbz, formyl, and the like), and —SO₂NRR, where R ishydrogen or alkyl.

“Substituted aryloxyaryl” refers to aryloxyaryl groups substituted withfrom 1 to 3 substituents on either or both aryl rings selected from thegroup consisting of hydroxy, acyl, acylamino, thiocarbonylamino,acyloxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, amidino,alkylamidino, thioamidino, amino, aminoacyl, aminocarbonyloxy,aminocarbonylamino, aminothiocarbonylamino, aryl, substituted aryl,aryloxy, substituted aryloxy, cycloalkoxy, substituted cycloalkoxy,heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy, substitutedheterocyclyloxy, carboxyl, carboxylalkyl, carboxyl-substituted alkyl,carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl, carboxylaryl,carboxyl-substituted aryl, carboxylheteroaryl, carboxyl-substitutedheteroaryl, carboxylheterocyclic, carboxyl-substituted heterocyclic,carboxylamido, cyano, thiol, thioalkyl, substituted thioalkyl, thioaryl,substituted thioaryl, thioheteroaryl, substituted thioheteroaryl,thiocycloalkyl, substituted thiocycloalkyl, thioheterocyclic,substituted thioheterocyclic, cycloalkyl, substituted cycloalkyl,guanidino, guanidinosulfone, halo, nitro, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy,substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy,heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino,oxythiocarbonylamino, —S(O)₂-alkyl, —S(O)₂-substituted alkyl,—S(O)₂-cycloalkyl, —S(O)₂-substituted cycloalkyl, —S(O)₂-alkenyl,—S(O)₂-substituted alkenyl, —S(O)₂-aryl, —S(O)₂-substituted aryl,—S(O)₂-heteroaryl, —S(O)₂-substituted heteroaryl, —S(O)₂-heterocyclic,—S(O)₂-substituted heterocyclic, —OS(O)₂-alkyl, —OS(O)₂-substitutedalkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NRR, —NRS(O)₂-alkyl,—NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl, —NRS(O)₂-substituted aryl,—NRS(O)₂-heteroaryl, —NRS(O)₂-substituted heteroaryl,—NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic,—NRS(O)₂—NR-substituted heterocyclic, mono- and di-alkylamino, mono- anddi-(substituted alkyl)amino, mono- and di-arylamino, mono- anddi-(substituted aryl)amino, mono and di-heteroarylamino, mono- anddi-(substituted heteroaryl)amino, mono- and di-heterocyclic amino, mono-and di-(substituted heterocyclic) amino, unsymmetric di-substitutedamines having different substituents selected from the group consistingof alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic, aminogroups on the substituted aryl blocked by conventional blocking groupsand substituted with —SO₂NRR, where R is hydrogen or alkyl.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 8 carbon atomshaving a single cyclic ring including, by way of example, cyclopropyl,cyclobutyl, cyclopentyl, cyclooctyl and the like. Excluded from thisdefinition are multi-ring alkyl groups such as adamantanyl.“Cycloalkenyl” refers to cyclic alkenyl groups of from 3 to 8 carbonatoms having single or multiple unsaturation but which are not aromatic.

“Substituted-cycloalkyl” and “substituted cycloalkenyl” refer to acycloalkyl and cycloalkenyl groups, preferably of from 3 to 8 carbonatoms, having from 1 to 5 substituents selected from the groupconsisting of oxo (═O), thioxo (═S), alkoxy, substituted alkoxy, acyl,acylamino, thiocarbonylamino, acyloxy, amino, amidino, alkylamidino,thioamidino, aminoacyl, aminocarbonylamino, aminothiocarbonylamino,aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryloxy,aryloxyaryl, substituted aryloxyaryl, halogen, hydroxyl, cyano, nitro,carboxyl, carboxylalkyl, carboxyl-substituted alkyl,carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl, carboxylaryl,carboxyl-substituted aryl, carboxylheteroaryl, carboxyl-substitutedheteroaryl, carboxylheterocyclic, carboxylsubstituted heterocyclic,cycloalkyl, substituted cycloalkyl, guanidino, guanidinosulfone, thiol,thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl,thiocycloalkyl, substituted thiocycloalkyl, thioheteroaryl, substitutedthioheteroaryl, thioheterocyclic, substituted thioheterocyclic,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkoxy, substituted cycloalkoxy, heteroaryloxy,substituted heteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy,oxycarbonylamino, oxythiocarbonylamino, —OS(O)₂-alkyl,—OS(O)₂-substituted alkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl,—OS(O)₂-heteroaryl, —OS(O)₂-substituted heteroaryl,—OS(O)₂-heterocyclic, —OS(O)₂-substituted heterocyclic, —OSO₂—NRR,—NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl,—NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl, —NRS(O)₂-substitutedheteroaryl, —NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic,—NRS(O)₂—NR-substituted heterocyclic, mono- and di-alkylamino, mono- anddi-(substituted alkyl)amino, mono and di-arylamino, mono- anddi-(substituted aryl)amino, mono- and di-heteroarylamino, mono- anddi-(substituted heteroaryl)amino, mono- and di-heterocyclic amino, mono-and di-(substituted heterocyclic) amino, unsymmetric di-substitutedamines having different substituents selected from the group consistingof alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic,substituted alkynyl groups having amino groups blocked by conventionalblocking groups (such as Boc, Cbz, formyl, and the like) andalkynyl/substituted alkynyl groups substituted with —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substituted alkenyl,—SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-aryl,—SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substituted heteroaryl,—SO₂-heterocyclic, —SO₂-substituted heterocyclic or —SO₂NRR, where R ishydrogen or alkyl.

“Cycloalkoxy” refers to —O-cycloalkyl groups. “Substituted cycloalkoxy”refers to —O-substituted cycloalkyl groups.

“Halo” refers to fluoro, chloro, bromo and iodo and preferably is eitherchloro or bromo.

“Heteroaryl” refers to an aromatic carbocyclic group of from 2 to 10carbon atoms and 1 to 4 heteroatoms selected from the groups consistingof oxygen, nitrogen and sulfur within the ring. Such heteroaryl groupscan have a single ring (e.g., pyridyl or furyl) or multiple condensedrings (e.g., indolizinyl or benzothienyl). Preferred heteroaryls includepyridyl, pyrrolyl, indolyl and furyl.

“Substituted heteroaryl” refers to heteroaryl groups which aresubstituted with from 1 to 3 substituents selected from the groupconsisting of hydroxy, acyl, acylamino, thiocarbonylamino, acyloxy,alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, amidino,alkylamidino, thioamidino, amino, aminoacyl, aminocarbonyloxy,aminocarbonylamino, aminothiocarbonylamino, aryl, substituted aryl,aryloxy, substituted aryloxy, cycloalkoxy, substituted cycloalkoxy,heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy, substitutedheterocyclyloxy, carboxyl, carboxylalkyl, carboxyl-substituted alkyl,carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl, carboxylaryl,carboxyl-substituted aryl, carboxylheteroaryl, carboxyl-substitutedheteroaryl, carboxylheterocyclic, carboxyl-substituted heterocyclic,carboxylamido, cyano, thiol, thioalkyl, substituted thioalkyl, thioaryl,substituted thioaryl, thioheteroaryl, substituted thioheteroaryl,thiocycloalkyl, substituted thiocycloalkyl, thioheterocyclic,substituted thioheterocyclic, cycloalkyl, substituted cycloalkyl,guanidino, guanidinosulfone, halo, nitro, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy,substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy,heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino,oxythiocarbonylamino, —S(O)₂-alkyl, —S(O)₂-substituted alkyl,—S(O)₂-cycloalkyl, —S(O)₂-substituted cycloalkyl, —S(O)₂-alkenyl,—S(O)₂-substituted alkenyl, —S(O)₂-aryl, —S(O)₂-substituted aryl,—S(O)₂-heteroaryl, —S(O)₂-substituted heteroaryl, —S(O)₂-heterocyclic,—S(O)₂-substituted heterocyclic, —OS(O)₂-alkyl, —OS(O)₂-substitutedalkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NRR, —NRS(O)₂-alkyl,—NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl, —NRS(O)₂-substituted aryl,—NRS(O)₂-heteroaryl, —NRS(O)₂-substituted heteroaryl,—NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic,—NRS(O)₂—NR-substituted heterocyclic, mono- and di-alkylamino, mono- anddi-(substituted alkyl)amino, mono- and di-arylamino, mono- anddi-(substituted aryl)amino, mono- and di-heteroarylamino, mono- anddi-(substituted heteroaryl)amino, mono- and di-heterocyclic amino, mono-and di-(substituted heterocyclic) amino, unsymmetric di-substitutedamines having different substituents selected from the group consistingof alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic, aminogroups on the substituted aryl blocked by conventional blocking groups(such as Boc, Cbz, formyl, and the like), and —SO₂ NRR, where R ishydrogen or alkyl.

“Heteroaryloxy” refers to the group —O-heteroaryl and “substitutedheteroaryloxy” refers to the group —O-substituted heteroaryl.

“Heterocycle” or “heterocyclic” refers to a saturated or unsaturatedgroup having a single ring or multiple condensed rings, containing from1 to 10 carbon atoms and from 1 to 4 heteroatoms selected from the groupconsisting of nitrogen, sulfur or oxygen within the ring. In fused ringsystems, one or more of the rings can be aryl or heteroaryl.

“Substituted heterocyclic” refers to heterocycle groups which aresubstituted with from 1 to 3 substituents selected from the groupconsisting of oxo (═O), thioxo (═S), alkoxy, substituted alkoxy, acyl,acylamino, thiocarbonylamino, acyloxy, amino, amidino, alkylamidino,thioamidino, aminoacyl, aminocarbonylamino, aminothiocarbonylamino,aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryloxy,aryloxyaryl, substituted aryloxyaryl, halogen, hydroxyl, cyano, nitro,carboxyl, carboxylalkyl, carboxyl-substituted alkyl,carboxyl-cycloalkyl, carboxyl-substituted cycloakyl, carboxylaryl,carboxyl-substituted aryl, carboxylheteroaryl, carboxyl-substitutedheteroaryl, carboxylheterocyclic, carboxylsubstituted heterocyclic,cycloalkyl, substituted cycloalkyl, guanidino, guanidinosulfone, thiol,thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl,thiocycloalkyl, substituted thiocycloalkyl, thioheteroaryl, substitutedthioheteroaryl, thioheterocyclic, substituted thioheterocyclic,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkoxy, substituted cycloalkoxy, heteroaryloxy,substituted heteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy,oxycarbonylamino, oxythiocarbonylamino, —OS(O)₂-alkyl,—OS(O)₂-substituted substituted alkyl, —OS(O)₂-aryl, —OS(O)₂-substitutedaryl, —OS(O)₂-heteroaryl, —OS(O)₂-substituted heteroaryl,—OS(O)₂-heterocyclic, —OS(O)₂-substituted heterocyclic, —OSO₂—NRR,—NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl,—NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl, —NRS(O)₂-substitutedheteroaryl, —NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-alkyl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic,—NRS(O)₂—NR-substituted heterocyclic, mono- and di-alkylamino, mono- anddi-(substituted alkyl)amino, mono-and di-arylamino, mono- anddi-(substituted aryl)amino, mono- and di-heteroarylamino, mono- anddi-(substituted heteroaryl)amino, mono- and di-heterocyclic mono- anddi-(substituted heterocyclic) amino, unsymmetric di-substituted amineshaving different substituents selected from the group consisting ofalkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic,substituted alkynyl groups having amino groups blocked by conventionalblocking groups and alkynyl/substituted alkynyl groups substituted with—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substitutedalkenyl, —SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-aryl,—SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substituted heteroaryl,—SO₂-heterocyclic, —SO₂-substituted heterocyclic or —SO₂NRR, where R ishydrogen or alkyl.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholino, thiomorpholino, piperidinyl, pyrrolidine,tetrahydrofuranyl, and the like.

“Heterocyclyloxy” refers to the group —O-heterocyclic, and “substitutedheterocyclyloxy” refers to the group —O-substituted heterocyclic.

The details of one or more implementations of the invention are setforth in the description below. Other features, objects, and advantagesof the invention will be apparent from the description and drawings, andfrom the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the nucleotide sequence of HLA-B*1502, including Regions1-6.

FIG. 2 shows the nucleotide sequence of HLA-B*5801, including Regions1-6.

FIG. 3 is a schematic diagram illustrating Competitive Sequence SpecificOligonucleotide-ELISA assay (CSSO-ELISA).

DETAILED DESCRIPTION OF THE INVENTION

Some evidence suggests that the pathogenesis of several similarmultisystem ADRs involves MHC-restricted presentation of a drug or itsmetabolites, which either directly bind to MHC molecules or bind toendogenous proteins, and activation of T cells (Svensson et al.,Pharmacol. Rev., 53(3):357-379, 2000). Skin-infiltrating CD830 cytotoxicT cells were found to be dominant in the bullous reactions such asSJS/TEN (Hari et al., Clin. Exp. Allergy, 31(9):1398-1408, 2001),whereas CD4+ helper T cells were characteristic of milder cutaneousADRs, such as maculopapular rash (Pichler et al., Int. Arch. AllergyImmunol., 113(1-3):177-180, 1997).

It was discovered that HLA-B*1502, HLA-B*5801, and HLA-B*4601 arerespectively associated with SJS/TEN induced by CBZ, with SJS/TEN/HSSinduced by allopurinol, and with milder cutaneous ADRs (e.g.,maculopapular rash, erythema multiforme, urticaria, or fixed drugeruption) induced by CBZ. Thus, these HLA-B alleles are useful geneticmarkers for determining whether a patient is at risk for developing ADRs(e.g., SJS, TEN, HSS, or milder cutaneous ADRs) induced by CBZcompounds, allopurinol compounds, or drugs otherwise having similarstructures thereof.

CBZ, also known as Tegretol, Tegol, G-32883, Biston, Calepsin,Carbatrol, Epitol, Finlepsin, Sirtal, Stazepine, Telesmin, or Timonil,is an aromatic anticonvulsant. Other aromatic anticonvulsants, includingphenytoin (Dilantin) and phenobarbital, cause similar ADRs as CBZ.Therefore, HLA-B*1502 can be employed to assess the risk for ADRs tothese other aromatic anticonvulsants as well. The aromaticanticonvulsants for which HLA-B*1502 can be used as a risk factor alsoinclude compounds or metabolites of CBZ, phenytoin or phenobarbital.Metabolites of these drugs are known in the art (see, e.g., Gennis etal., 1991; Leeder, Epilepsia, 39 Suppl. 7:S8-16, 1998; Naisbitt et al.,Mol. Pharmacol., 63(3):732-741, 2003), such as CBZ-10,11 epoxide,CBZ-10,11-diol, CBZ-2,3-diol, dihydro CBZ, CBZ catechol and CBZo-quinone, p-hydroxy phenytoin, phenytoin dihydrodiol, phenytoincatechol, phenytoin methylcatechol, and phenytoin o-quinone.

Allopurinol is a drug for hyperuricemia and chronic gout.

The present invention provides a method of predicting whether a patientis at risk for developing ADRs, particularly SJS, TEN, or HSS, based onthe presence of certain HLA alleles or their equivalent genetic markersin that patient.

In one example, the presence of HLA-B*1502 in a patient indicates thatthe patient is at risk for developing SJS/TEN induced by CBZ compounds,compounds otherwise structurally similar to CBZ, or metabolites thereof.Table I shows examples of compounds that can induce SJS/TEN in anHLA-B*1502 carrier.

TABLE I Drug compounds associated with SJS in patients with HLA-B* 1502Names of active ingredients or the Brand Name Structures of the activeingredient carbamazepine, Epitol, Tegretol, Microtrol Bipotrol,carbamazepine, carbamazepine, Pharmavene, carbamazepine, Carbatrol, SPD-417 5H-Dibenz[b,f]azepine-5- carboxamide [CAS]

Oxcarbazepine, Trileptal GP-47680, GP-47779, GP-47779 MHD, KIN-493,oxacarbazepine, TRI-476, TRI-477, TRI-477 (MHD), Trileptal NP, [CAS]:5H-Dibenz[b,f]azepine-5- carboxamide, 10,11-dihydro- 10-oxo

licarbazepine BIA-2-093 BIA-2-005 [CAS]: (S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenzo/b,f/azepine- 5-carboxamide

modafinil, Sparlon(New Formulation drug), [CAS]: Acetamide, 2-[(diphenylmethyl)sulfinyl]-

In another example, the presence of HLA-B*5801 in a patient indicatesthat the patient is at risk for SJS/TEN or HSS induced by allopurinolcompounds, compounds otherwise structurally similar to allopurinol, ormetabolites thereof. Table II shows examples of compounds that caninduce SJS/TEN or HSS in patients carrying HLA-B*5801.

TABLE II Drug compounds associated with SJS in patients carrying HLA-B*5801 Names Structure of the active ingredient Allopurinol, Aloprim,Zyloprim, Apo- Allopurinol, Purinol

Oxypurinol, Oxyprim [CAS]: 1H-Pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

Febuxostat, TEI-6720, TMX-67 [CAS]: 5-Thiazolecarboxylic acid,2-[3-cyano-4-(2- methylpropoxy)phenyl]- 4-methyl-

Y-700 1-(3-Cyano-4- neopentyloxyphenyl)pyrazole- 4-carboxylic acid

modafinil, Sparlon(New Formulation drug), [CAS]: Acetamide, 2-[(diphenylmethyl)sulfinyl]-

In yet another example, the presence of HLA-B*4601 in a patientindicates that the patient is at risk for developing mild cutaneousADRs, e.g., maculopapular rash, induced by CBZ.

Other HLA-B alleles can also be predispositive for cutaneous ADRs. Forexample, ankylosing spondylitis is strongly associated with HLA-B27alleles, such as B*2701-B*2723. (Khan, Curr. Opin. Rheumatol.,12(4):235-238, 2000; Feltkamp et al., Curr. Opin. Rheumatol.,13(4):285-290, 2001).

The presence of a genetic marker (e.g., an HLA allele) can be determinedby direct detection of that marker or particular regions within it.Genomic DNAs for allele detection can be prepared from a patient bymethods well known in the art, e.g., PUREGENE DNA purification systemfrom Gentra Systems, Minnesota. Detection of a region within a geneticmarker of interest includes examining the nucleotide(s) located ateither the sense or the anti-sense strand within that region. Methodsknown in the art can be used to detect a particular region, e.g.,Sequence specific oligonucleotides-hybridization, Real-time PCR, orCSSO-ELISA (M. Hiratsuka et al, J. of Biochemical and Biophysic.Methods, 67:87-94, 2006).

The presence of HLA-B*1502 can be determined by detecting at least twoof Regions 1-6 shown in FIG. 1. The presence of these regions can bedetermined by detecting nucleotides at certain positions within theseregions, e.g., positions 1 and 3 in Region 1, positions 1 and 6 inRegion 2, and positions 1 and 3 in Region 3. Presence of any two ofRegions 1-6 indicates that the patient is an HLA-B*1502 carrier.

The presence of HLA-B*5801 can be determined by detecting at least twoof Regions 1-6 shown in FIG. 2. The presence of these regions can bedetermined by detecting the nucleotides at certain positions withinthese regions, e.g., positions 1, 2, and 5 in Region 1, positions 1, 4,6, 7, 8, 15, 16, and 20 in Region 2, positions 2, 4, 5, 8, and 9 inRegion 3, positions 3 and 5 in Region 4, positions 1 and 9 in Region 5,and positions 3 and 10 in Region 6. Presence of any two of Regions 1-6indicates that the patient is an HLA-B*5801 carrier.

The DNA products obtained from PCR can be detected usingsequence-specific probes, e.g., hydrolysis probes from TaqMan, Beacons,Scorpions; or hybridization probes These probes are designed such thatthey bind to the regions of interest, e.g., Regions 1-6 of HLA-B*1502 orRegions 1-6 of HLA-B*5801. The PCR products also can be detected byDNA-binding agents, e.g., SYBR® Green.

The presence of an allele of interest also can be determined bydetecting genetic markers equivalent to the allele. Genetic markers nearthe allele of interest tend to co-segregate, or show a linkagedisequilibrium, with the allele. Consequently, the presence of thesemarkers (equivalent genetic markers) is indicative of the presence ofthe allele of interest, which, in turn, is indicative of a risk for ADRdevelopment. Exemplary genetic markers equivalent to HLA-B*1502 includeDRB1*1202, Cw*0801, Cw*0806, A*1101, and MICA*019. Exemplary markersequivalent to HLA-B*5801 include A*3303, Cw*0302, DRB1*0301, andMICA*00201.

An equivalent genetic marker can be of any type, e.g., an HLA allele, amicrosatellite marker, or a single nucleotide polymorphism (SNP) marker.A useful equivalent genetic marker is normally about 200 kb or less(e.g., 100 kb, 80 kb, 60 kb, 40 kb, or 20 kb) from the allele ofinterest. Methods described above or known in the art can be used todetect the presence or absence of an equivalent genetic marker.

Alternatively or in addition, RNAs, cDNAs, or protein products ofalleles of interest can be detected to determine the presence or absenceof the allele. For example, serotyping or microcytotoxity methods can beused to determine the protein product of an HLA allele.

To further increase the accuracy of risk prediction, the allele ofinterest and/or its equivalent genetic marker can be determined alongwith the genetic markers of accessory molecules and co-stimulatorymolecules which are involved in the interaction betweenantigen-presenting cells and T-cells. These genetic markers includemicrosatellite and single nucleotide polymorphism (SNP) markers. Theaccessory and co-stimulatory molecules include cell surface molecules(e.g., CD80, CD86, CD28, CD4, CD8, T cell receptor (TCR), ICAM-1, CD11a,CD58, CD2, etc.) and inflammatory or pro-inflammatory cytokines,chemokines (e.g., TNF-α), and mediators (e.g., complements, apoptosisproteins, enzymes, extracellular matrix components, etc.). Also ofinterest are genetic markers of drug metabolizing enzymes which areinvolved in the bioactivation or detoxification of drugs. These geneticmarkers also include microsatellite and SNP markers. The drugmetabolizing enzymes include phase I enzymes (e.g., cytochrome P450superfamily etc.) and phase II enzymes (e.g., microsomal epoxidehydrolase, arylamine N-acetyltransferase, UDP-glucuronosyl-transferase,etc.).

The present invention further provides a method for pharmacogenomicprofiling. A panel of genetic factors is determined for a givenindividual, and each genetic factor is associated with thepredisposition for a disease or medical condition, including ADRs. Inthe present method, the panel of genetic factors includes at least oneallele selected from the group consisting of HLA-B*1502, HLA-B*5801 andHLA-B*4601. The panel can include two alleles or all three alleles fromthe group. In addition to HLA-B*1502, 5801 and/or 4601, the panel caninclude any other known genetic factors, such as thiopurinemethyltransferase and genes for the long-QT syndrome. The geneticmarkers for accessory molecules, co-stimulatory molecules and/or drugmetabolizing enzymes described above can also be included.

Also within the scope of the invention is a kit containing probes fordetecting genetic markers, e.g., HLA-B*1502, HLA-B*5801 or HLA-B*4601.The term “probe” used herein refers to any substance useful fordetecting another substance. Thus, a probe can be an oligonucleotide orconjugated oligonucleotide that specifically hybridizes to a particularregion within an allele of interest. The conjugated oligonucleotiderefers to an oligonucleotide covalently bound to chromophore or amolecules containing a ligand (e.g., an antigen), which is highlyspecific to a receptor molecular (e.g., an antibody specific to theantigen). The probe can also be a PCR primer, together with anotherprimer, for amplifying a particular region within the allele ofinterest. Further, the probe can be an antibody that recognizes anallele of interest or a protein product of the allele. Optionally, thekit can contain a probe that targets an internal control allele, whichcan be any allele presented in the general population, e.g. GAPDH,β-actin, KIR. Detection of an internal control allele is designed toassure the performance of the kit.

The kit can further include tools and/or reagents for collectingbiological samples from patients, as well as those for preparing genomicDNA, cDNAs, RNAs or proteins from the samples. For example, PCR primersfor amplifying the relevant regions of the genomic DNA may be included.The kit can also contain probes for genetic factors useful inpharmacogenomic profiling, e.g., thiopurine methyltransferase.

In one example, the kit contains a first probe and a second probe, eachfor detecting a region selected from Regions 1-6 of HLA-B*1502 or fromRegions 1-6 of HLA-B*5801. The first and second probes target differentregions. These two probes can be a pair of PCR primers, or labeledoligonucleotides useful in hybridization assays. Optionally, the kit caninclude a third probe for detecting an internal control allele. It canalso include additional probes for detecting additional regions withinHLA-B*1502 or HLA-B*5801.

In yet another aspect, this invention provides a method of identifying adrug compound that induces an ADR (e.g., SJS/TEN or HSS) in a patientcarrying an HLA allele associated with the ADR (e.g., HLA-B*1502,HLA-B*5801 or HLA-B*4601). It is suggested that drugs can be presentedby certain HLA complexes to activate T lymphocytes, consequentlyinducing ADRs. T cells reactive to these drugs are suggested to beinvolved in the development of ADRs induced by these drugs. Thus,compounds that can activate these T cells are candidates for inducingADRs in a patient carrying one or more HLA alleles associated with theseADRs. As a result, this method can be used as a screening method in newdrug development to find out drug compounds that could induce such ADRs.

Genotyping can be performed on an ADR patient to determine whether thepatient carries an HLA allele associated with the disease. T lymphocytesand antigen-presenting cells (e.g., B cells or monocytes) can beisolated from the patient and cultured in vitro following methods wellknown in the art. (Naisbitt D J, Mol Pharmacol. 2003 March; 63(3):732-41, Wu et al, J Allergy Clin Immunol. 2006 July; 118(1):233-41.E-published on 2006 Apr. 27). B cells so isolated can be transformed byEpstein-Bar virus to generate B cell lines. T cells reactive to a drugcan be expanded in the presence of both the drug and autologousantigen-presenting cells. The expanded T cells can then be exposed to atest compound in the presence of autologous antigen-presenting cells todetermine whether the test compound can activate the T cells. If so, thetest compound is a candidate that can induce the ADR in a patientcarrying the same HLA allele.

Without further elaboration, it is believed that the above descriptionhas adequately enabled the present invention. The following examplesare, therefore, to be construed as merely illustrative, and notlimitative of the remainder of the disclosure in any way whatsoever. Allof the publications cited herein are hereby incorporated by reference intheir entirety.

Example 1 Detection of HLA-B*1502 and HLA-B*5801 Using Real-Time PCR

Genomic DNAs were extracted from a patient's blood or saliva. Threepairs of PCR primers each targeting Regions 1 and 5, Regions 1 and 4, orRegions 1, 3 and 4 of HLA-B*1502 (see FIG. 1) were synthesized. Inaddition, one pair of primers targeting Regions 2 and 4 of HLA-B*5801(see FIG. 2) were synthesized. The targeted regions were amplified anddetected using SYBR® Green Real-Time PCR system (Applied Biosystem).Briefly, the primers, the genomic DNAs, and the Power SYBR® Green PCRmaster mixture (included in the real-time PCR system) were mixedtogether and the PCR was carried out by: (i) activating polymerase at95° C. for 10 minutes, (ii) denaturing at 95° C. for 15 seconds andannealing/elongating DNA chains at 71° C. for 1 minute, (iii) conducting40 cycles of denaturing/annealing/elongating, and (iv) disassociatingthe amplified product from its template at 95° C. for 15 seconds, and60° C. for 1 minute. PCR amplification of a killer-cellimmunoglobulin-like receptor (KIR) was applied as an internal control.The presence or absence of HLA-B*1502 or HLA-B*5801 in a patient wasdetermined based on the Ct value of HLA-B*1502 or HLA-B*5801 and thedifference of Ct values (ΔCt value) between HLA-B*1502/HLA-B*5801 andKIR. The Ct value (threshold cycle) is determined by identifying thecycle number at which the reporter dye emission intensity is abovebackground noise. The threshold cycle is determined at the mostexponential phase of the reaction and is more reliable than end-pointmeasurements of accumulated PCR products used by traditional PCRmethods. The threshold cycle is inversely proportional to the copynumber of the target template, the greater the template number, thelower the threshold cycle measured.

170 genomic DNA samples extracted from human B cell lines and 35 genomicDNA samples prepared from human blood or saliva were tested fordetecting the presence of HLA-B*1502 following the method describedabove. The Ct values of HLA-B*1502 and KIR were in the range of 23-27and 19-25, respectively. The HLA-B*1502 were recognized as positive whenthe range of ΔCt between HLA-B*1502 and KIR was smaller than 7. In these170 supercontrol, 15 gDNA with HLA-B*1502 were present and were verifiedby Dynal SSO kits, and the results indicate that both the sensitivityand specificity of this method reach >99%.

170 genomic DNA samples extracted from human B cell lines and 87 genomicDNA samples prepared from human blood or saliva were tested fordetecting the presence of HLA-B*5801 following the method describedabove. For DNA samples derived from HLA-B*5801 positive patients, the Ctvalues of HLA-B*5801 and KIR were in the range of 22-28 and 10-26,respectively. The HLA-B*5801 were recognized as positive when the rangeof ΔCt between HLA-B*5801 and KIR was smaller than 7. For DNA samplesderived from HLA-B*5801 negative patients, the Ct value of HLA-B*5801was about 34 and the ΔCt was greater than 13. In all the samples, 51gDNA were found HLA-B*5801 positive and were verified by Dynal SSO kits.These results indicate that both the sensitivity and specificity reached>99%.

Example 2 Detection of HLA-B*1502 and HLA-B*5801 Using CSSO-ELISA

The procedures for carrying out CSSO-ELISA are outlined in FIG. 3.

In general, using genomic DNAs as templates, PCRs were conducted toproduce products which contain the specific regions shown in FIG. 1 orFIG. 2. Either the Forward primer or the Reversed Primer was labeledwith a Ligand I (LI), which was recognizable by the Molecular I linkedwith an enzyme (e.g. HRP) The PCR reactions were designed and conductedto produce the products containing one or more specific regions, i.e.,Regions 1-6 of HLA-B*1502 or Regions 1-6 of HLA-B*5801. Two competitivesequence specific oligonucleotides (CSSO1 and CSSO2) were designed.CSSO1 specifically recognizes one of Regions 1-6 of HLA-B*1502 orRegions 1-6 of HLA-B*5801, and CSSO2 was designed to prime to the commontype of HLA-B*15 (i.e. non-B*1502 alleles or non-B*-5801. The CSSO1 waslabeled with Ligand 2, which is recognizable by Molecular II coated ontoa reaction plateor strip. The PCR products thus obtained were hybridizedwith the two CSSOs on the reaction plate or a strip. After washing awayany free molecule, substrate of the enzyme was added to the reactionplate. The enzymatic reaction, signaled by the presence of a color, isindicative of the presence of HLA-B*1502 or HLA-B*5801.

Example 3 Correlation between CBZ-Induced SJS/TEN and HLA-B*1502

A total of 238 ADR patients (Mongoloids or Mongoloid descendents) wererecruited either from Chang Gung Memorial Hospital or from several othermedical centers throughout Taiwan for this study. Their drug-takinghistory including dosage and duration, and the phenotypes of ADRs wererecorded. The diagnostic criteria of clinical morphology were definedaccording to Roujeau, J. Invest Dermatol., 102(6):28S-30S, 1994. Forexample, SJS was defined as skin detachment of less than 10% ofbody-surface area, overlap SJS-TEN as skin detachment of 10-30%, and TENas greater than 30%. SJS, overlap SJS-TEN and TEN are collectivelyreferred to as SJS/TEN.

For each patient, the suspected drug was withdrawn and the patientobserved for symptoms. Patients who developed a cutaneous ADR that didnot subside upon withdrawal of the drug were excluded.

According to the criteria described above, 112 patients were diagnosedwith SJS/TEN and 126 patients had a milder hypersensitivity reaction tovarious drugs. Among the 112 SJS/TEN patients, 42 were exposed to CBZ(tegretol), 17 had taken allopurinol, and 53 were under medicationsother than CBZ and allopurinol.

73 tegretol-tolerant patients were included as controls. Volunteers fromthe general population of Taiwanese (n=94; age range: 20 to 80 years)were also recruited. The study was approved by the institutional reviewboard, and informed consent was obtained.

All of these patients were subjected to genotyping. Briefly, reagentsfor performing a reverse line blot assay using sequence-specificoligonucleotide (SSO) were purchased from DYNAL Biotech Ltd.(Bromborough, UK). PCR products were generated using biotinylatedprimers for the second and the third exons of the HLA class I or classII loci, and then hybridized to a line blot of SSO of probes immobilizedon a nylon membrane. The presence of biotinylated PCR product bound to aspecific probe is detected using streptavidin-horseradish peroxidase(HRP) and a chromogenic, soluble substrate to produce a blue “line” atthe position of the positive probe. The probe reactivity pattern wasinterpreted by the genotyping software Dynal RELI™ SSO (DYNAL BiotechLtd.; Bromborough, UK). Potential ambiguities were further resolved bysequence-based typing and DNA sequencing performed according to the IHWGTechnical Manual (International Histocompatibility Working Group), seeGenomic Analysis of the Human MHC DNA Based Typing for HLA Alleles andLinked Polymorphisms. Marcel G. J. Tilanus, Editor in Chief, ISBN No.0-945278-02-0.

To some patients, SNP genotyping was performed using high throughputMALDI-TOF mass spectrometry. Briefly, primers and probes were designedusing the SpectroDESIGNER software (Sequenom, San Diego, Calif., USA).Multiplex polymerase chain reactions (PCR) were performed, theunincorporated dNTPs were dephosphorylated using the shrimp alkalinephosphatase (Hoffman-LaRoche, Basel, Switzerland), followed by primerextension. The purified primer extension reaction was spotted onto a384-element silicon chip (SpectroCHIP, Sequenom), analyzed using aBruker Biflex III MALDI-TOF SpectroREADER mass spectrometer (Sequenom)and spectra processed with SpectroTYPER (Sequenom).

Allele frequencies in the different groups were compared by theChi-square method with Yates correction by constructing 2×2 tables. Pvalues were corrected for comparisons of multiple HLA alleles (Pc) bymultiplying the raw P values by the observed number of HLA allelespresent within the loci. Odds ratios were calculated with Haldane'smodification, which adds 0.5 to all cells to accommodate possible zerocounts.

As shown in Table 1, a DNA variant allele in the HLA-B locus(HLA-B*1502) was associated in patients with drug-induced SJS/TEN,particularly in patients receiving CBZ (tegretol).

TABLE 1 HLA-B*1502 frequency in 42 Taiwanese patients having CBZ-inducedSJS/TEN Patients Controls1^(a) Controls2^(b) Controls3^(c) Odds Allele N= 42 N = 142 N = 94 N = 73 X² Ratio P_(c) B*1502 42 (100%) 9 (6.3)%137.28 1194.47  3.6 × 10⁻³⁰ B*1502 42 (100%) 5 (5.3%) 110.919 1383.22.15 × 10⁻²⁴ B*1502 42 (100%) 3 (4.1%) 98.936 1712  9.1 × 10⁻²²^(a)patients who had milder ADRs other than SJS ^(b)general Taiwanesepopulation ^(c)patients who are CBZ-tolerant X², Chi-square with Yatescorrection P_(c), calculated by multiplying the raw P values by theobserved number of HLA-B alleles (35).

HLA-B*1502 was detected in 42 of 42 (100%) SJS/TEN patients who receivedCBZ. The allele was also found in 17 of 53 (32%) SJS/TEN patients whoreceived other drugs (8 phenytoin, 2 allopurinol, 2 amoxicillin, 1sulfasalazine, 1 ketoprofen, 1 Ibuprofen, and 2 unknown drugs).Particularly, 8 of 17 patients (47.05%) who developed SJS/TEN aftertaking phenytoin also carried the HLA-B*1502 allele. On the other hand,the allele was only found in 4.1% (3/73) of the CBZ-tolerant group, 0%(0/32) of the phenytoin-tolerant group, 6.3% (9/142) of the patients whohad milder ADRs other than SJS, and 5.3% (5/94) of the generalpopulation. By using the tolerant group as a control, the odds ratio,sensitivity, specificity, positive predictive value, and negativepredictive value for B*1502 associated CBZ-induced SJS/TEN, were 1712,100%, 95.89%, 96.0%, and 100%, respectively. With such a high predictivevalue and sensitivity, typing of this HLA-B allele can be used inidentifying high-risk patients for drug-induced SJS/TEN, particularlyCBZ or phenytoin induced SJS/TEN.

The mild ADRs induced by CBZ was found to be associated with anotherallele, HLA-B*4601. 10 out of 16 (62.5%) of the patients with thesemilder reactions to CBZ had HLA-B*4601. In contrast, the allele was onlyfound in 26% (19/73) of the CBZ-tolerant group. The odds ratio forB*4601 associated CBZ-induced milder cutaneous ADRs was 4.73.Consequently, HLA-B*4601 can be used in the risk assessment for mildcutaneous ADR induced by CBZ.

TABLE 2 Phenotype/genotype data of patients having CBZ-induced cutaneousADRs ID Suspected drug Phenotype HLA-B Genotype 1 Carbamazepine SJSB*1502/B*3802 2 Carbamazepine SJS B*1502/B*3501 3 Carbamazepine SJSB*1502/B*4006 4 Carbamazepine SJS B*1502/B*3802 5 Carbamazepine SJSB*1502/B*3802 6 carbamazepine, SJS B*1502/B*3802 phenytoin 7Carbamazepine SJS B*1502/B*4001 8 Carbamazepine SJS B*1502/B*3901 9Carbamazepine SJS B*1502/B*5801 10 Carbamazepine SJS B*1502/B*5801 11Carbamazepine SJS B*1502/B*1525 12 Carbamazepine SJS B*1502/B*4002 13Carbamazepine SJS B*1502/B*4006 14 Carbamazepine SJS B*1502/B*5801 15Carbamazepine Overlap SJS/TEN B*1301/B*1502 16 Carbamazepine OverlapSJS/TEN B*1502/B*3501 17 Carbamazepine SJS B*1502/B*3802 18Carbamazepine SJS B*1502/B*4601 19 Carbamazepine SJS B*1301/B*1502 20Carbamazepine SJS B*1502/B*5801 21 Carbamazepine SJS B*1502/B*4601 22Carbamazepine, SJS B*1502 NSAID 23 Carbamazepine SJS B*1502/B*3501 24Carbamazepine SJS B*1502/B*4601 25 Carbamazepine SJS B*1502/B*4601 26Carbamazepine SJS B*1502/B*5801 27 Carbamazepine SJS B*1501/B*1502 28Carbamazepine SJS B*1502/B*4001 29 Carbamazepine SJS B*1502 30carbamazepine, SJS B*1502/B*5801 meloxicam, sulidanc, phenytoin 31Carbamazepine SJS B*1502/4601 32 Carbamazepine SJS B*1502/5801 33Carbamazepine SJS B*1502/4601 34 Carbamazepine SJS B*1502/5502 35Carbamazepine SJS B*1502 36 carbamazepine, SJS B*1502/4002 phenytoin 37Carbamazepine SJS B*1502/4001 38 Carbamazepine SJS B*1502 39carbamazepine, SJS B*1502 phenytoin 40 Carbamazepine Overlap SJS/TENB*1502/4001 41 Carbamazepine Overlap SJS/TEN B*1502/4601 42Carbamazepine SJS B*1502/3802 43 Carbamazepine maculopapular rashB*5801/B*4601 44 Carbamazepine erythema multiform B*4001/B*4601 45Carbamazepine maculopapular rash B*1301/B*4001 46 Carbamazepine Andangioedema B*4601/B*5401 47 Carbamazepine maculopapular rashB*4001/B*4601 48 Carbamazepine, maculopapular rash B*4001/B*4001 NSAID49 Carbamazepine maculopapular rash B*1301/B*5502 50 Carbamazepine lipswelling, oral and B*4601/B*5801 genital ulcer 51 CarbamazepineMaculopapular B*4601/B*5801 52 Carbamazepine And angioedema B*4001 53Carbamazepine maculopapular rash B*4001/B*5101 54 Carbamazepinemaculopapular rash B*1301/4001 55 Carbamazepine maculopapular rashB*4001/B*4601 56 Carbamazepine erythema multiform B*4601/B*5401 57Carbamazepine maculopapular rash B*4601 58 Carbamazepine erythemamultiform B*4601/5101

Example 4 Correlation between Allopurinol-Induced SJS/TEN and HLA-B*5801

HLA-B*5801 was found to be associated with allopurinol-induced SJS/TEN.This HLA-B allele was found in all 17 (100%) SJS/severe ADR patientstreated allopurinol (Tables 3 and 4), but was found in only 18% of thegeneral Taiwanese population (odds ratio 155, sensitivity 100%,specificity 82%, positive predictive value 84.7%, negative predictivevalue 100%, Pc=3.7×10⁻⁹). These results suggest that HLA-B*5801 is auseful genetic marker, either alone or in combination with other geneticmarkers, for assessing whether a patient taking allopurinol is at riskfor developing SJS/TEN.

TABLE 3 HLA-B*5801 frequency in 17 Taiwanese patients withallopurinol-induced severe cutaneous ADRs Allele Patients n = 17Controls1^(a) n = 142 Controls2^(b) n = 94 X² odds ratio P_(c) B*5801 17(100%) 26(18.3%) 47.2 153.86 2.1 × 10⁻¹⁰ B*5801 17 (100%) 17 (18.0%)41.7 155 3.7 × 10⁻⁹  ^(a)patients who had ADRs other thanallopurinol-induced cutaneous ADR ^(b)general Taiwanese population X²,Chi-square with Yates correction P_(c), calculated by multiplying theraw P values by the observed number of HLA-B alleles (35).

TABLE 4 Phenotype/genotype data of patients with allopurinol-inducedcutaneous ADRs Patient ID Suspected drug Phenotype HLA-B Genotype 59allopurinol SJS B*0705/B*5801 60 allopurinol SJS B*4001/B*5801 61allopurinol SJS B*1554/B*5801 62 allopurinol SJS B*3901/B*5801 63allopurinol SJS B*5801 64 allopurinol SJS B*3901/B*5801 65 allopurinolSJS B*3901/B*5801 66 allopurinol SJS B*4001/B*5801 67 Allopurinol SJSB*1502/B*5801 68 allopurinol SJS B*4001/B*5801 69 allopurinol SJS andvasculitis on leg B*4601/B*5801 70 allopurinol SJS, and lichenoidB*4001/B*5801 71 allopurinol SJS B*4002/B*5801 72 allopurinol SJSB*4001/B*5801 73 alloprinol SJS B*5101/B*5801 74 allopurinol TENB*1301/5801 75 alloprinol SJS B*5801

Example 5 Correlation between HLA-B*5801 and Allopurinol-Induced HSS

HLA-B*5801 was also found to be linked to allopurinol-induced HSS, whichincludes cutaneous rash (e.g., diffuse macuopapular, exfoliativedermatitis), fever, eosinophilia, atypical circulating lymphocytes,leukocytosis, acute hepatocellular injury, or worsening renal function(Arellano et al., Ann. Pharmacother., 27:337, 1993).

31 patients were studied, among which 21 had SJS, 3 SJS/TEN, 1 TEN, and15 HSS. In all enrolled cases, allopurinol was regarded as the offendingdrug if the onset of ADR syndromes occurred within the first 2 months ofallopurinol exposure and the ADRs symptoms disappeared upon withdrawalof the drug. Patients with any of the following conditions wereexcluded: absence of symptoms after re-exposure to allopurinol, andpatients with milder skin eruption who did not meet the criteria of HSS,SJS or TEN.

The onset of HSS symptoms for all of the 31 patients was within thefirst 2 months of allopurinol exposure and 2 patients had a secondattack within 2 days of re-exposure to allopurinol. Twelve patientsreceived other drug(s) in addition to allopurinol, but their medicalrecords revealed no ADRs when these concomitant medications were takenwithout allopurinol. All patients had hyperuricemia and/or goutyarthritis, as well as other chronic illnesses, including hypertension(14/31), chronic renal disease (16/31), and diabetes (9/31).

Ninety-eight gouty arthritis patients who had been on allopurinol for atleast 6 months (mean=38 months, range=6-107 months) with no syndromes ofADRs were included as the allopurinol-tolerant control. The sexdistribution of tolerant group is comparable to general prevalence ofgout in Chinese people. Furthermore, 93 normal subjects served as thenormal control group. The demographic variables of these 3 groups areshown in Table 5.

TABLE 5 Demographic variables, dosage and duration of allopurinolexposure in severe ADRs patients, tolerant patients, as well as normalsubjects Normal Severe ADRs Tolerant Subjects (n = 31) (n = 98) (n = 93)Sex Male 12 89 52 Female 19  9 41 Age (years) Median (range) 57.9(18-91) 57.3 (21-84)  53.9 (22-91) Allopurinol dosage (mg/day) Median(range) 143.3 (50-300) 159.2 (100-400) None Duration of allopurinolexposure Median (range) 28.2 days (1-56) 38 months (6-107) None

HLA-B*5801 allele was present in all 31 (100%) of the patients havingallopurinol-induced severe ADRs, in 16 (16.3%) of the 98allopurinol-tolerant patients (odds ratio 315, Pc<10⁻¹⁵), and in 19(20%) of the 93 normal subjects (odds ratio 241, Pc<10⁻¹³). Relative tothe allopurinol-tolerant group, the absence of HLA-B*5801 had a negativepredictive value of 100% for allopurinol-induced ADRs, and the presenceof this allele had a positive predictive value of 66%. Accordingly,HLA-B*5801 is a useful marker with high specificity (84%) andsensitivity (100%) for allopurinol-induced severe ADRs, includingcutaneous ADRs (e.g., SJS/TEN or HSS) and allopurinol-induced DRESS(drug reaction with eosinophilia and systemic symptoms).

Example 6 Genetic Markers Equivalent to HLA-B*1502 or HLA-B*5801

Genetic markers near an HLA allele of interest tend to co-segregate, orshow a linkage disequilibrium, with the allele of interest. As a result,the presence of these markers (equivalent genetic markers) is indicativeof the presence of the allele.

To test the incidence of potential equivalent genetic markers inpatients with ADRs, several markers in the HLA-B*1502 haplotype weredetermined for their association with ADRs. Indeed, HLA markers of theHLA-B*1502 haplotype, such as DRB1*1202, Cw*0801, Cw*0806, A*1101, andMICA*019, had a significantly high frequency in SJS/TEN patients who hadbeen exposed to CBZ (Table 6).

Markers associated with HLA-B*5801 were also determined. Alleledistribution was analyzed in 4 patients who were homozygous forHLA-B*5801 and the ancestral haplotype of this allele was defined asincluding HLA-A*3303, Cw*0302, B*5801 and DRB1*0301. This ancestralhaplotype was presented in 12 (38.7%) of the 31 allopurinol-ADRspatients (Table 7), but only in 7.1% of the tolerant patients and 9.7%of the normal subjects.

TABLE 6 Correlation between markers of B*1502- ancestral haplotypes andADRs CBZ CBZ CBZ Allopurinol General SJS/TEN Milder Tolerant SJS/TENPopulation (n = 42) (n = 16) (n = 73) (n = 17) (n = 94) HLA-B*1502 42(100%) 0 (0%) 3 (4.1%) 1 (5.8%) 5 (5.3%) HLA-Cw*0801 38 (90%) ND 10(13.7%) 2 (11.7%) 10 (10.6%) HLA-Cw*0806 3 (7.1%) ND 0 (0%) 0 (0%) 0(0%) HLA-A*1101 31 (73.8%) ND ND ND 28 (29.8%) HLA-DRB1*1202 35 (83.3%)ND ND ND 19 (20.2%)

TABLE 7 Frequencies of individual or combined loci of HLA-B*5801ancestral haplotype in patients with allopurinol-induced severe ADRs,allopurinol-tolerant patients, and in normal subjects Allopurinol-Allopurinol- Normal ADRs tolerant Subjects (n = 31) (n = 98) (n = 93)B*5801 31 (100%)   16 (16.3%)¹  19 (20.4%)² Cw*0302 29 (93.5%) 15(15.3%) 19 (20.4%) A*3303 20 (64.5%) 18 (18.4%) 20 (21.5%) DRB1*0301 21(67.7%) 14 (14.3%) 14 (15.1%) B*5801, Cw*0302 29 (93.5%) 15 (15.3%) 19(20.4%) B*5801, Cw*0302, A*3303 20 (64.5%) 13 (13.3%) 16 (17.2%) B*5801,Cw*0302, 19 (61.3%) 9 (9.2%) 10 (10.8%) DRB1*0301 B*5801, Cw*0302,A*3303, 12 (38.7%) 7 (7.1%) 9 (9.7%) DRB1*0301 ¹Odds ratio(Allopurinol-ADRs/Tolerant): 315 (95% Cl, 18.3-5409.5), p_(c) = 7.5 ×10⁻¹⁶. ²Odds ratio (Allopurinol-ADRs/Normal): 241 (95% Cl, 14.1-4111),p_(c) = 6.1 × 10⁻¹⁴.

MHC markers associated with HLA-B*5801 were also determined using shorttandem repeat polymorphism assay (STRP). Briefly, twenty highlypolymorphic microsatellite markers located in the MHC region wereselected from NCBI database (i.e., D6S258, D6S2972, D6S510, D6S265,D6S388, D6S2814, HLAC_CA1, HLABC_CA2, MIB, MICA, TNFd, BAT2_CA, D6S273,D6S1615, DQCAR, G51152, D6S2414, D6S1867, D6S1560, and D6S1583). Theaverage heterozygosity of these markers was 0.72 with an estimatedspacing of 230 kb.

Primers were designed based on the sequences of these markers describedin the database. PCRs were carried out to amplify and detect thepresence or absence of these markers in patients using GeneAmp 9700thermocyclers (Applied Biosystems, Foster City, Calif., USA) (in a 5-μlvolume containing 10 ng of genomic DNA and 0.33 μM of each primer). Upto 6 PCR products having appropriate sizes and displaying fluorescentsignals were pooled before capillary gel electrophoresis. The size ofpolymorphic amplicons was determined by electrophoresis of ABI 3730 DNAsequencer (Applied Biosystems), using the LIZ500 size standard as aninternal size standard (Applied Biosystems). Allele sizing wascalculated using the GENMAPPER program version 3.0 (Applied Biosystems).Allele calling and binning were performed using the SAS program. ThreeCEPH control individuals (1331-01, 1331-02, 1347-2) and H20 wereincluded in all genotyping experiments for quality control purposes.

An allele block located between HLA-C and TNFd was found in theallopurinol-induced ADR patient group, but not in theallopurinol-tolerant group, using a linkage disequilibrium plot. In thisblock, a haplotype (MIB*358-MICA*206-TNFd*140) near the HLA-B allele wasidentified. The association of this haplotype with ADRs is consistentwith the association of HLA-B*5801 with the same ADRs (p=0.0018). Byusing STRP markers and sequencing of the MICA allele, allallopurinol-induced ADR patients were found to carry the same B allele(B*5801), MICA allele (MICA*00201) and TNF STRP marker (TNFd*140).Except for one patient, all others were also found to carry the same MIBmarker (MIB*358).

Example 7 Cross-Reactivity of CBZ-Reactive T Cells to Oxcarbazepine andLicarbazepine

Two patients having CBZ-induced SJS/TEN were recruited from Chang GungMemorial Hospital. One of the patients carried HLA-B*1502/B*4601, theother HLA-B*1502/B*5101. Genomic DNAs were extracted from the patientsusing PUREGENE DNA purification system (Gentra systems, Minnesota, USA).The HLA-B alleles were verified using sequence-specific oligonucleotidereverse line blots (DYNAL Biotech Ltd., Bromborough, U.K.).

Peripheral blood mononuclear cells (PBMCs) were isolated from thepatients by Ficoll-Isopaque (Pharmacia Fine Chemicals, Piscataway, N.J.)density gradient centrifugation. A portion of the PBMCs were transformedby Epstein-Barr virus to establish autologous B-cell lines.

T cells reactive to CBZ were expanded as described below. PBMCs preparedfrom the patients were cultured in complete RPMI medium containing 10%heat-inactivated human serum, IL-2 (25 U/ml), and CBZ (25 μ/ml) (Sigma)in a 37° C., 5% CO₂ incubator for 7 days. The T cells were then expandedby co-culturing with irradiated (50 Gy) autologous B cells in thepresence of CBZ for 10 days. After 2 cycles of the above co-culturingprocedure, the CBZ-activated T cells were collected and subjected toELISPOT assays (eBioscience).

The CBZ-reactive T cells were tested for their cross-reactivity tocompounds e.g., CBZ 10,11-epoxide, Oxcarbazepine (brand name:trileptal), Licarbazepine, and sunlindac. Briefly, T lymphocytes (5×10³cells) were mixed with autologous B cells (5×10⁴ cells) in 200 μl RPMImedium containing 10% FBS in the presence or absence of a test compound.The cells were then incubated for 24 hours in the wells of an ELISPOTplate coated with anti-interferon γ antibodies (Millipore). Afterincubation, the supernatant of the cell culture was collected andinterferon-γ contained therein was detected using antibody-mediatedmethods known in the art.

Results from this study indicate that CBZ-reactive T cells werecross-reactive to CBZ 10,11-epoxide, Oxcarbazepine, and Licarbazepine,but not to Sulindac.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the scope of thefollowing claims.

1-20. (canceled)
 21. A method of assessing a risk of a human patient fordeveloping an adverse drug reaction in response to a drug, comprisingdetecting the presence of HLA-B*1502 in a sample obtained from thepatient, and correlating the presence of HLA-B*1502 in the sample withan increased risk for an adverse drug reaction in the patient inresponse to oxcarbazepine, wherein the adverse drug reaction isStevens-Johnson syndrome or toxic epidermal necrolysis.
 22. The methodof claim 21, wherein the sample obtained from the patient is a DNAsample.
 23. The method of claim 22, wherein the presence of the HLAallele is determined by hybridization with an oligonucleotide thatspecifically hybridizes to the allele.
 24. The method of claim 22,wherein the DNA sample is obtained from peripheral blood, saliva, urine,or hair of the patient.
 25. The method of claim 21, wherein the sampleobtained from the patient is a RNA sample, a protein sample, a cellsample, or a serum sample.
 26. The method of claim 25, wherein thesample is obtained from peripheral blood of the patient.
 27. The methodof claim 21, wherein the adverse drug reaction is Stevens-Johnsonsyndrome.
 28. The method of claim 21, wherein the adverse drug reactionis toxic epidermal necrolysis.